Lerner, I., Bartok, O., Wolfson, V., Menet, J. S., Weissbein, U., Afik, S., Haimovich, D., Gafni, C., Friedman, N., Rosbash, M. and Kadener, S. (2015). Clk post-transcriptional control denoises circadian transcription both temporally and spatially. Nat Commun 6: 7056. PubMed ID: 25952406Summary:
The transcription factor CLOCK (CLK) is essential for the development and maintenance of circadian rhythms in Drosophila. However, little is known about how CLK levels are controlled. This study shows that Clk mRNA is strongly regulated post-transcriptionally through its 3' UTR. Flies expressing Clk transgenes without normal 3' UTR exhibit variable CLK-driven transcription and circadian behaviour as well as ectopic expression of CLK-target genes in the brain. In these flies, the number of the key circadian neurons differs stochastically between individuals and within the two hemispheres of the same brain. Moreover, flies carrying Clk transgenes with deletions in the binding sites for the miRNA bantam have stochastic number of pacemaker neurons, suggesting that this miRNA mediates the deterministic expression of CLK. Overall these results demonstrate a key role of Clk post-transcriptional control in stabilizing circadian transcription, which is essential for proper development and maintenance of circadian rhythms in Drosophila.

Chouhan, N.S., Wolf, R., Helfrich-Förster, C. and Heisenberg, M. (2015).Flies remember the time of day. Curr Biol [Epub ahead of print]. PubMed
ID: 26028434Summary:
The circadian clock enables organisms to anticipate daily environmental
cycles and drives corresponding changes in behavior. Such endogenous
oscillators also enable animals to display time-specific memory. For
instance, mice and honeybees associate the location of a stimulus (like
food or mate) with a certain time of day (time-place learning). However,
the mechanism underlying time-related learning and memory is not known.
The present study investigated time-specific odor learning
by using a genetically tractable animal, the fly Drosophila
melanogaster. Starved flies were trained in the morning and
afternoon to associate distinct odors with sucrose reward. The training
was repeated the next day, and their time-dependent odor preference was
tested on the third day. Drosophila were found to
express appetitive memory at the relevant time of day if the two
conditioning events were separated by more than 4 hr. Flies could form
time-odor associations in constant darkness (DD) as well as in a daily
light-dark (LD) cycle, but not when kept under constant light (LL)
conditions. Circadian clock mutants, period01
(per01) and clockAR
(clkAR), learned to associate sucrose reward with a certain odor
but were unable to form time-odor associations. The findings of this study
show that flies can utilize temporal information as an additional cue in
appetitive learning. Time-odor learning in flies depends on a per-
and clk-dependent endogenous mechanism that is independent of
environmental light cues.

Liu, T., Mahesh, G., Houl, J. H. and Hardin, P. E. (2015). Circadian activators are expressed days before they initiate clock function in late pacemaker neurons from Drosophila. J Neurosci 35: 8662-8671. PubMed ID: 26041931Summary:Circadian pacemaker neurons in the Drosophila brain control daily rhythms in locomotor activity. These pacemaker neurons can be subdivided into early or late groups depending on whether rhythms in period (per) and timeless (tim) expression are initiated at the first instar (L1) larval stage or during metamorphosis, respectively. Because CLOCK-CYCLE (CLK-CYC) heterodimers initiate circadian oscillator function by activating per and tim transcription, a Clk-GFP transgene was used to mark when late pacemaker neurons begin to develop. It was surprising to see that CLK-GFP was already expressed in four of five clusters of late pacemaker neurons during the third instar (L3) larval stage. CLK-GFP is only detected in postmitotic neurons from L3 larvae, suggesting that these four late pacemaker neuron clusters are formed before the L3 larval stage. A GFP-cyc transgene was used to show that CYC, like CLK, is also expressed exclusively in pacemaker neurons from L3 larval brains, demonstrating that CLK-CYC is not sufficient to activate per and tim in late pacemaker neurons at the L3 larval stage. These results suggest that most late pacemaker neurons develop days before novel factors activate circadian oscillator function during metamorphosis.

Lee, E., Cho, E., Kang, D. H., Jeong, E. H., Chen, Z., Yoo, S. H. and Kim, E. Y. (2016). Pacemaker-neuron-dependent disturbance of the molecular clockwork by a Drosophila CLOCK mutant homologous to the mouse Clock mutation. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27489346Summary:Circadian clocks are composed of transcriptional/translational feedback loops (TTFLs) at the cellular level. In Drosophila TTFLs, the transcription factor Clock (Clk)/Cycle (Cyc) activates clock target gene expression, which is repressed by the physical interaction with Period (Per). This study shows that amino acids (AA) 657-707 of Clk, a region that is homologous to the mouse Clock exon 19-encoded region, is crucial for Per binding and E-box-dependent transactivation in S2 cells. Consistently, in transgenic flies expressing Clk with an AA657-707 deletion in the Clock (Clkout) genetic background (p{Clk-Δ};Clkout), oscillation of core clock genes' mRNAs displayed diminished amplitude compared with control flies, and the highly abundant dCLK&Delta657-707 showed significantly decreased binding to Per. Behaviorally, the p{dClk-Δ};Clkout flies exhibited arrhythmic locomotor behavior in the photic entrainment condition but showed anticipatory activities of temperature transition and improved free-running rhythms in the temperature entrainment condition. Surprisingly, p{dClk-Δ};Clkout flies showed pacemaker-neuron-dependent alterations in molecular rhythms; the abundance of dCLK target clock proteins was reduced in ventral lateral neurons (LNvs) but not in dorsal neurons (DNs) in both entrainment conditions. In p{dClk-Delta};Clkout flies, however, strong but delayed molecular oscillations in temperature cycle-sensitive pacemaker neurons, such as DN1s and DN2s, were correlated with delayed anticipatory activities of temperature transition. Taken together, this study reveals that the LNv molecular clockwork is more sensitive than the clockwork of DNs to dysregulation of dCLK by AA657-707 deletion. Therefore, it is proposed that the dCLK/CYC-controlled TTFL operates differently in subsets of pacemaker neurons, which may contribute to their specific functions.

Cho, E., Lee, E. and Kim, E. Y. (2016). Diversification of molecular clockwork for tissue specific function: insight from novel Drosophila Clock mutant homologous to mouse Clock allele. BMB Rep 49(11):587-589. PubMed ID: 27756446Summary:
The circadian clock system enables organisms to anticipate the rhythmic environmental changes and to manifest behavior and physiology at advantageous times of day. Transcriptional/translational feedback loop (TTFL) is the basic feature of eukaryotic circadian clock and is based on the rhythmic association of circadian transcriptional activator and repressor. In Drosophila, repression of dCLOCK/CYCLE (dCLK/CYC) mediated transcription by PERIOD (PER) is critical for inducing circadian rhythms of gene expression. Pacemaker neurons in the brain control specific circadian behaviors upon environmental timing cues such as light and temperature cycle. This study shows here that amino acids 657-707 of dCLK is important for the transcriptional activation and the association with PER both in vitro and in vivo. Flies expressing dCLK lacking AA657-707 in Clkout genetic background, homologous to the mouse Clock allele where exon 19 region is deleted, display pacemaker-neuron-dependent perturbation of the molecular clockwork. Namely, the molecular rhythms in light-cycle-sensitive pacemaker neurons such as ventral lateral neurons (LNvs) were significantly disrupted but those in temperature-cycle-sensitive pacemaker neurons such as dorsal neurons (DNs) were robust. These results suggest that the dCLK-controlled TTFL diversified in pacemaker-neuron-dependent manner which contribute to specific functions such as different sensitivities to entraining cues.

Vaccaro, A., Issa, A. R., Seugnet, L., Birman, S. and Klarsfeld, A. (2017). Drosophila Clock is required in brain pacemaker neurons to prevent premature locomotor aging independently of its circadian function. PLoS Genet 13(1): e1006507. PubMed ID: 28072817Summary:Circadian clocks control many self-sustained rhythms in physiology and behavior with approximately 24-hour periodicity. This study examined the effects of clock disruptions on locomotor aging and longevity in Drosophila. Lifespan was found to be similarly reduced in three arrhythmic mutants (ClkAR, cyc0 and tim0) and in wild-type flies under constant light, which stops the clock. In contrast, ClkAR mutants showed significantly faster age-related locomotor deficits (as monitored by startle-induced climbing) than cyc0 and tim0, or than control flies under constant light. Clk, but not Cyc, inactivation by RNA interference in the pigment-dispersing factor (PDF)-expressing central pacemaker neurons led to similar loss of climbing performance as ClkAR. Conversely, restoring Clk function in these cells was sufficient to rescue the ClkAR locomotor phenotype, independently of behavioral rhythmicity. Accelerated locomotor decline of the ClkAR mutant required expression of the PDF receptor and correlated to an apparent loss of dopaminergic neurons in the posterior protocerebral lateral 1 (PPL1) clusters. This neuronal loss was rescued when the ClkAR mutation was placed in an apoptosis-deficient background. Impairing dopamine synthesis in a single pair of PPL1 neurons that innervate the mushroom bodies accelerated locomotor decline in otherwise wild-type flies. These results therefore reveal a novel circadian-independent requirement for Clk in brain circadian neurons to maintain a subset of dopaminergic cells and avoid premature locomotor aging in Drosophila.

Nippe, O. M., Wade, A. R., Elliott, C. J. H. and Chawla, S. (2017). Circadian rhythms in visual responsiveness in the behaviorally arrhythmic Drosophila clock mutant Clk(Jrk). J Biol Rhythms 32(6):583-592. PubMed ID: 29172879Summary:
An organism's biological day is characterized by a pattern of anticipatory physiological and behavioral changes that are governed by circadian clocks to align with the 24-h cycling environment. This study used flash electroretinograms (ERGs) and steady-state visually evoked potentials (SSVEPs) to examine how visual responsiveness in wild-type Drosophila melanogaster and the circadian clock mutant ClkJrk varies over circadian time. The ERG parameters of wild-type flies vary over the circadian day, with a higher luminance response during the subjective night. The SSVEP response that assesses contrast sensitivity also showed a time-of-day dependence, including 2 prominent peaks within a 24-h period and a maximal response at the end of the subjective day, indicating a tradeoff between luminance and contrast sensitivity. Moreover, the behaviorally arrhythmic ClkJrk mutants maintained a circadian profile in both luminance and contrast sensitivity, but unlike the wild-types, which show bimodal profiles in their visual response, ClkJrk flies show a weakening of the bimodal character, with visual responsiveness tending to peak once a day. It is concluded that the ClkJrk mutation mainly affects 1 of 2 functionally coupled oscillators and that the visual system is partially separated from the locomotor circadian circuits that drive bouts of morning and evening activity. As light exposure is a major mechanism for entrainment, this work suggests that a detailed temporal analysis of electrophysiological responses is warranted to better identify the time window at which circadian rhythms are most receptive to light-induced phase shifting.

Liu, T., Mahesh, G., Yu, W. and Hardin, P. E. (2017). CLOCK stabilizes CYCLE to initiate clock function in Drosophila. Proc Natl Acad Sci U S A 114(41): 10972-10977. PubMed ID: 28973907Summary:
The Drosophila circadian clock keeps time via transcriptional feedback loops. These feedback loops are initiated by CLOCK-CYCLE (CLK-CYC) heterodimers, which activate transcription of genes encoding the feedback repressors PERIOD and TIMELESS. Circadian clocks normally operate in approximately 150 brain pacemaker neurons and in many peripheral tissues in the head and body, but can also be induced by expressing CLK in nonclock cells. These ectopic clocks also require cyc, yet CYC expression is restricted to canonical clock cells despite evidence that cyc mRNA is widely expressed. This study shows that CLK binds to and stabilizes CYC in cell culture and in nonclock cells in vivo. Ectopic clocks also require the blue light photoreceptor CRYPTOCHROME (CRY), which is required for both light entrainment and clock function in peripheral tissues. These experiments define the genetic architecture required to initiate circadian clock function in Drosophila, reveal mechanisms governing circadian activator stability that are conserved in perhaps all eukaryotes, and suggest that Clk, cyc, and cry expression is sufficient to drive clock expression in naive cells.

Du, J., Zhang, Y., Xue, Y., Zhao, X., Zhao, X., Wei, Y., Li, Z., Zhang, Y. and Zhao, Z. (2018). Diurnal protein oscillation profiles in Drosophila head. FEBS Lett. PubMed ID: 30311939Summary:Circadian clocks control daily rhythms in physiology, metabolism, and behavior in most organisms. Proteome-wide analysis of protein oscillations is still lacking in Drosophila. In this study, the total protein and phosphorylated protein in Drosophila heads in a 24-hour daily time-course were assayed by using the iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) method, and 10 and 7 oscillating proteins as well as 19 and 22 oscillating phosphoproteins in the w(1118) wild type and Clk(Jrk) mutant strains were separately identified. Lastly, a mini screen was performed to investigate the functions of some oscillating proteins in circadian locomotion rhythms. This study provides the first proteomic profiling of diurnally oscillating proteins in fly heads, thereby providing a basis for further mechanistic studies of these proteins in circadian rhythm.

In Drosophila, there are two well-characterized photoperiod response genes: period (per) and timeless (tim). The protein levels, RNA levels, and transcription rates of these two genes undergo robust circadian oscillations. In addition, mutations in the two proteins (Per and Tim) alter or abolish the periodicity and phase of these rhythms, demonstrating that both proteins regulate their own transcription. Although there is no evidence indicating that the effects on transcription are direct, Per contains a PAS domain, which has been shown to mediate interactions between transcription factors. Most of these PAS-containing transcription factors also contain well-characterized basic helix-loop-helix (bHLH) DNA-binding domains. However, Per lacks any known DNA-binding domain, and there is no evidence that Per interacts directly with DNA. Therefore it was proposed that Per regulates transcription by interacting with DNA-binding transcription factors of the bHLH-PAS family and how Per transcription is regulated remained an open question (Allada, 1998 and references).

Recent data have extended this model in two ways: (1) an enhancer has been identified in the per promoter capable of driving cycling transcription of a reporter gene (Hao, 1997). Notably, the activity of this 69-base pair element requires an E box (CACGTG), a known binding site for some bHLH transcription factors, including bHLH-PAS transcription factors. (2) The cloning of the mouse circadian rhythm gene, mClock, revealed a bHLH-PAS transcription factor involved in circadian rhythms. Recently, mouse per genes have been identified and found to undergo circadian oscillation in mammalian clock tissues. Thus, mouse CLOCK may drive the cycling transcription of mouse per genes through evolutionarily conserved E box elements in mouse per promoters. If so, one might expect to find a Drosophila orthologs of mClock, which would drive cycling of the Drosophila per gene (Allada, 1998).

Jrk, a novel arrhythmic Drosophila mutant, has been identified which severely disrupts cycling transcription of the per and tim genes. The cloning and identification of the Jrk gene reveals that it is the apparent homolog of the mouse Clock gene; it has therefore been named Drosophila Clock (Clk). The mutation in Clk results in a premature stop codon that truncates the protein, deleting most of the putative C-terminal activation domain. This truncation is consistent with the semidominant mutant phenotype, similar to the original mouse Clock mutant (Allada, 1998).

A second Drosophila clock gene, cycle (cyc) has also been cloned. Homozygous mutant cyc flies have a behavioral and molecular phenotype that resembles closely that of homozygous Clk flies: they are behaviorally arrhythmic and exhibit little per and tim transcription. Further phenogenetic analyses indicate that, like Clk, the cyc locus has a dosage effect on period. It is suggested that cycle is a nonvital, dedicated clock gene. Cloning of cyc indicates that, like Clk, it encodes a bHLH-PAS transcription factor and is a Drosophila homolog of the human gene BMAL1 (MOP3) (Ikeda, 1997; Hogenesch, 1997 and 1998). Biochemical work (Hogenesch, 1998) indicates that BMAL1 is the partner of mammalian CLOCK and that the heterodimer binds to and activates transcription from per-like E boxes. Based on all of these results, it is proposed that the CYC:CLK heterodimer binds to per and tim E boxes and makes a major contribution to the circadian transcription of Drosophila clock genes (Rutila, 1998). Further characterization of the Drosophila Clk mutant phenotype and a second study (Hogenesch, 1998) suggest that the wild-type Drosophila protein (Clk) interacts directly with the per and tim E boxes and makes a major contribution to the circadian transcription of clock genes. The similar mouse mutant phenotype and the remarkable sequence conservation strongly support the presence of similar clock mechanisms and components in the common ancestor of Drosophila and mammals more than 500 million years ago (Allada, 1998).

Placed in the context of the current understanding of the Drosophila circadian oscillator, these results indicate that Clock closes the feedback loop. A Clock-Cycle complex drives expression of per and tim by binding an E-box that is present in their promoters. With time, Per and Tim heterodimers accumulate, translocate to the nucleus, and act as dominant negative inhibitors of Clock-Cycle. As mRNA and protein levels fall, the inhibition is relieved, which allows Clock-Cycle to initiate a new round of synthesis (Darlington, 1998).

Drosophila Clock (Clk) is rhythmically expressed, with peaks in mRNA and protein (Clk) abundance early in the morning. Clk mRNA cycling is regulated by Period-Timeless (Per-Tim)-mediated release of Clk- and Cycle (Cyc)-dependent repression. Lack of both Per-Tim
derepression and Clk-Cyc repression results in high levels of Clk mRNA, which implies that a separate Clk activator is present. These results
demonstrate that the Drosophila circadian feedback loop is composed of two interlocked negative feedback loops: a per-tim loop, which is activated by
Clk-Cyc and repressed by Per-Tim, and a Clk loop, which is repressed by Clk-Cyc and derepressed by Per-Tim (Glossop, 1999).

Comparatively little is known about the regulation of Clk
mRNA cycling. The levels of Clk mRNA are low in mutants
lacking Per (per01) or Tim
(tim01) function, which suggests that Per and
Tim activate Clk transcription in addition to their roles
as transcriptional repressors. The mechanism of
Per-Tim-dependent activation is not known, but three models have been
proposed to account for this activation. In the
first two models, Per and Tim promote Clk transcription by
shuttling transcriptional activators into the nucleus or by coactivating a transcriptional complex. In the third model, Per or Tim or both inhibit the
activity of a transcriptional repressor complex (Glossop, 1999).

To distinguish among these alternative models,
Clk mRNA levels were measured in different clock gene mutant
combinations. Because Clk and Cyc are both required for per
and tim activation, it was predicted that mutants lacking
functional Clk (ClkJrk) or Cyc
(Cyc0) would exhibit low levels of
Clk mRNA because the concentrations of the Per and Tim
activators (of Clk) would be low. It was surprising to find
that the level of Clk mRNA is indistinguishable from the
wild-type peak in both mutants.
The levels of Clk mRNA do not vary significantly over
the circadian cycle in these mutants, which
is consistent with the lack of a functional circadian oscillator (Glossop, 1999).

The high level of Clk mRNA in the absence of Clk-dependent
Per accumulation indicates that Per-dependent Clk
activation does not occur by nuclear localization of an activator or by
coactivation. However, the possibility remains that low levels of per and tim transcripts in
ClkJrk or Cyc0 mutants lead to some active Per-Tim dimer
formation and subsequent activation of Clk transcription. To eliminate this possibility, Clk mRNA levels were measured in per01;ClkJrk and
per01;Cyc0 double
mutants. In both cases, the levels of Clk mRNA observed under light-dark (LD) or constant dark (DD) conditions are close to
the peak level in wild-type flies, indicating that Per-Tim activates
Clk transcription through derepression (Glossop, 1999).

The Clk repressor that is removed as a result of Per-Tim
accumulation appears to be either Clk-Cyc itself or a repressor that
is activated by Clk-Cyc. When comparing the levels of Clk between per01 flies and
per01;ClkJrk or
per01;Cyc0 double
mutants, the presence of active Clk and Cyc results in the repression
of Clk transcript accumulation. In
per01 mutants, Clk mRNA is at low
but detectable levels. This suggests
that in the absence of Per-Tim derepression, Clk
transcription reaches a steady state in which activation and
Clk-Cyc-dependent repression equilibrate to produce low levels of
Clk mRNA transcripts and, hence, of Clk protein. In
per01 and tim01 mutants,
per and tim transcription is constitutive and
per and tim transcripts are relatively low in
abundance. This result can be
explained by the partial activation of per and
tim by low levels of Clk-Cyc dimers in the absence of
Per-Tim repression (Glossop, 1999).

On the basis of these observations, it is proposed that interlocked
negative feedback loops mediate circadian oscillator function in
Drosophila (see reviews by Hardin, The Circadian Timekeeping System of Drosophila and Vallone, Start the clock! Circadian rhythms and development). Late at
night, Per-Tim dimers in the nucleus bind to and sequester Clk-Cyc
dimers. This interaction effectively inhibits Clk-Cyc function, which
leads to the repression of per and tim
transcription and the derepression of Clk transcription. As
Per-Tim levels fall early in the morning (ZT 0-3), Clk-Cyc dimers are
released and repress Clk expression, thereby decreasing Clk mRNA levels so that they are low by the end of the day
(ZT 12). Concomitant with the drop in
Clk mRNA levels (through Clk-Cyc-dependent repression) is
the accumulation of per and tim mRNA (through
E-box-dependent Clk-Cyc activation). As
Clk mRNA falls to low levels early in the evening (ZT 15),
the levels of Clk-Cyc also fall, leading to a decrease
in per and tim transcription and an increase in
Clk mRNA accumulation. A new cycle then begins as high
levels of Per and Tim enter the nucleus and Clk starts to accumulate late at night (Glossop, 1999).

These observations also fit well with the regulation of
Drosophila cryptochrome (cry), whose mRNA cycles
in phase with that of Clk. Like
Clk, CRY mRNA transcripts are constitutively low
in per01 mutants and constitutively high in
ClkJrk or Cyc0 single
mutants and in
per01;ClkJrk or
per01;Cyc0 double mutants. These striking similarities between Clk
and CRY mRNA phases (in the wild type) and Clk
and CRYmRNA levels in circadian mutants suggest that the
cry locus may be regulated by the same Per-Tim release of
Clk-Cyc repression mechanism as Clk (Glossop, 1999).

These results reveal the existence of a Clk feedback loop
and its regulatory interactions with the well-characterized
per-tim feedback loop. One clear prediction from these
experiments is that there is a separate activator of Clk
expression. Such an activator is indicated by the high levels of
Clk mRNA in the absence of Per and of either Clk or Cyc.
This observation is somewhat surprising because the presence of this
activator is independent of factors that control the expression of
other clock genes (that is, Per, Clk, and Cyc) (Glossop, 1999).

Data supporting the existence of interlocked per-tim and
Clk feedback loops were obtained from whole heads, raising
the possibility that Clk expression in small subsets of
'clock-specific' cells such as the locomotor activity pacemaker
cells (that is, lateral neurons) could be masked by Clk expression in other tissues. However, the autonomy and
synchrony of per expression in diverse tissues in the head
and body suggest that the circadian feedback loop mechanism is the same
in all tissues and argue against fundamental
tissue-specific differences in the feedback loop mechanism (Glossop, 1999).

An important aspect of circadian biology is how the clock regulates
clock-controlled genes (CCGs). In mammals, it has been shown in vitro
that CLOCK and BMAL-1 (the mammalian ortholog of Cyc) activate
vasopressin gene transcription and that all three mouse Pers and Tim
repress this activation, resulting in peak vasopressin mRNA transcripts
by midmorning (ZT 6). Although this mode of
regulation may be more general for CCGs whose mRNA transcripts peak in
phase with per (or mPer), it does not explain how
CCGs that cycle in antiphase are regulated. The results presented here
provide a possible mechanism by which the clock regulates CCGs whose
mRNAs cycle in antiphase to those of per. The similarities
between Clk and cry mRNA profiles in the wild
type and in several single and double circadian mutants suggest that
Per-Tim release of Clk-Cyc repression may serve a more general role in
regulating CCG mRNAs that cycle in antiphase to per mRNA (Glossop, 1999).

Two distinct modes of PERIOD recruitment onto dCLOCK reveal a novel role for TIMELESS in circadian transcription

Negative transcriptional feedback loops are a core feature of eukaryotic circadian clocks and are based on rhythmic interactions between clock-specific repressors and transcription factors. In Drosophila, the repression of dClock (dClk)-Cycle (Cyc) transcriptional activity by dPeriod (dPer) is critical for driving circadian gene expression. Although growing lines of evidence indicate that circadian repressors such as dPer function, at least partly, as molecular bridges that facilitate timely interactions between other regulatory factors and core clock transcription factors, how dPer interacts with dClk-Cyc to promote repression is not known. This study identified a small conserved region on dPer required for binding to dClk, termed CBD (for dClk binding domain). In the absence of the CBD, dPer is unable to stably associate with dClk and inhibit the transcriptional activity of dClk-Cyc in a simplified cell culture system. CBD is situated in close proximity to a region that interacts with other regulatory factors such as the Doubletime kinase, suggesting that complex architectural constraints need to be met to assemble repressor complexes. Surprisingly, when dPer missing the CBD (dPerδCBD) was evaluated in flies the clock mechanism was operational, albeit with longer periods. Intriguingly, the interaction between dPerδCBD and dClk is Tim-dependent and modulated by light, revealing a novel and unanticipated in vivo role for Tim in circadian transcription. Finally, dPerδCBD does not provoke the daily hyperphosphorylation of dClk, indicating that direct interactions between dPer and dClk are necessary for the dClk phosphorylation program but are not required for other aspects of dClk regulation (Sun, 2010).

A shared feature of eukaryotic circadian pacemaker mechanisms is that daily cycles in gene expression involve the phase-specific interaction of one or more repressors with core clock transcription factors. Initial findings identified Per proteins in animals and Frequency (Frq) in Neurospora as key components underlying the main negative feedback loops operating in their respective clocks. Early models, mostly based on work in Drosophila and Neurospora, suggested that the direct binding of Per or Frq to their relevant transcription factors was the biochemical mode-of-action underlying these repressors. More recent work is beginning to refine this view and it is now thought that these 'repressors' function, at least partly, by acting as molecular bridges to ensure the timely assembly and/or delivery of larger repressor complexes that inhibit elements functioning in the positive arms of circadian transcriptional feedback loops. Intriguingly, Per and Frq proteins also share another role in that phosphorylation driven changes in their daily levels are central to setting clock pace. Thus, repressors such as Per and Frq act as a critical nexus in clock mechanisms by connecting phosphorylation-based biochemical timers to the regulation of transcription, yielding appropriately phased daily cycles in gene expression. How the binding of these period-setting repressors to core clock transcription factors leads to inhibition in transactivation potential is not well understood (Sun, 2010).

This study identified the C4 region on dPer as the sole or major dClk binding domain (termed CBD). Despite the inability of dPerδCBD to bind to dClk, p{dperδCBD} flies manifest quasi-normal feedback circuitry within the core oscillator mechanism, almost certainly as a result of Tim facilitating the close interaction of dPer with dClk, enabling temporal repression of dClk-Cyc-mediated transcription. Thus, Tim is a bona fide component of the in vivo circadian repressor complex, and presumably plays a role in modulating the interaction between dClk and dPer. Moreover, this study reports that direct binding between dClk and dPer is not necessary for dPer's repressor activity, but is likely necessary for normal hyperphosphorylation of dClk (Sun, 2010).

An approach that is providing insights into how these 'phospho-timing repressor' clock proteins function is the identity of regions required for promoting transcriptional inhibition. In Drosophila, early work mapped a region on dPer necessary for strong inhibition of dClk/Cyc activity in an S2 cell transcription assay. This region, termed the CCID domain, contains two highly conserved regions, namely C3 and C4. The C3 region contains the sole or major domain on dPer required for stable interaction with Dbt, termed the dPDBD, and is critical not only for hyperphosphorylation of dPer but also for inhibiting dClk-Cyc-mediated transcription, despite the fact that eliminating this region does not abrogate the ability of dPer to stably interact with dClk in vivo. In contrast, the newly identified CBD is required for the physical interaction of dPer with dClk. Thus, the CCID is comprised of at least two distinct regions with different biochemical modes-of-action; a region required for physical interaction with dClk and another that functions as a scaffold to promote binding of regulatory factors that modulate the activity/metabolism of dClk/Cyc (Sun, 2010).

An unanticipated aspect of this work is that tim can promote the binding of dPerδCBD to dClk in a simplified cell culture system and in flies, an event that rescues dPer's repressor function. Earlier work suggested that Tim is dispensable for repression of dClk/Cyc transactivity. However, although Tim exhibits little to no repressor activity toward dClk-Cyc-mediated transcription in S2 cells, it enhances that of dPer. This enhancement was largely attributed to the fact that Tim stimulates the nuclear localization of dPer. The current findings suggest a physiological role for Tim in modulating circadian transcription by regulating the interaction between dPer and dClk. Although dPer can bind dClk in the absence of Tim, it is possible that by interacting with both dPer and dClk, Tim influences the properties of the repressor complex. For example, Tim might modulate the conformation of dPer, enhancing the assembly/activity of the repressor complex. In support of this view, brief light stimulation in the night leads to subtle but noticeable effects on dper/tim RNA levels that precede significant changes in the abundance of dPer. Of particular interest, dper/tim RNA levels were induced by light exposure in flies expressing a dPer variant (named dPer-δC2) missing a short stretch of conserved amino acids (515-568). Although the basis for the impaired function of dPer-δC2 was not clear, based on the current results it is possible that its interaction with dClk/Cyc is defective in the absence of Tim (Sun, 2010).

The findings that Tim has a more pivotal role in transcriptional regulation might also be relevant to a recent study suggesting a two-step mechanism for dPer-mediated inhibition of dClk-Cyc activity, whereby during the first phase of inhibition dPer is bound to dClk at the chromatin, followed by a second off-DNA sequestration of dClk by dPer (Menet, 2010). The switch from an on-DNA to an off-DNA mechanism is thought to occur around ZT18, around the time tim begins to accumulate in the nucleus. It is speculated that Tim might regulate progression from an on-DNA to an off-DNA inhibitory mechanism by modulating the interaction between dPer and dClk/Cyc (Sun, 2010).

Although dPerδCBD in the presence of Tim can suppress dClk-Cyc-mediated transcription, the efficiency is lower compared with that of wild-type dPer. Several different scenarios could account for this, including less favorable spatial alignment between dPerδCBD and dClk/Cyc and/or effects on the ability of dPerδCBD to bind and/or deliver regulatory factors to dClk/Cyc. Perhaps a more interesting possibility is suggested by the less extensive phosphorylation of dClk in p{dperδCBD} flies. Hyperphosphorylated dClk is mainly detected in the late night/early day during times when dClk-Cyc transcriptional activity is inhibited, suggesting that highly phosphorylated dClk is less active. The absence of hyperphosphorylated isoforms of dClk in p{dperδCBD} flies suggests that direct association between dPer and dClk is required to provoke dPer-dependent dClk phosphorylation. The lack of hyperphosphorylated isoforms of dClk in p{dperδCBD} flies might also contribute to the higher overall levels of dper/tim transcripts. In this context it is noteworthy that Frq is thought to play a major role in repressing the positive limb of the circadian transcriptional circuits in Neurospora by regulating the phosphorylated state of the WCC complex, the key clock transcription factor driving cyclical gene expression in that system. Future studies will be required to better understand the biochemical function of dClk phosphorylation (Sun, 2010).

In summary, these findings demonstrate that the direct interaction of a key repressor to its target transcription factors is not necessary for its ability to engage in transcriptional inhibition. Moreover, Tim can promote the close association of dPer to dClk in a manner that sustains dPer's repressor capability, revealing a more direct role for tim in functional interactions between the negative and positive limbs of the circadian transcriptional feedback circuits operating in Drosophila. The dClk interaction domain on dPer is situated very close to the dPDBD region that functions, at least partly, by acting as a bridge to promote close interactions between regulatory factors (such as Dbt) and the dClk/Cyc complex. The close spacing on dPer between these two functional regions suggests that complex architectural constraints need to be met to assemble highly efficient repressor complexes. It will be of interest to determine whether other repressors, such as Per proteins in mammals and Frq in Neursopora, also have similar spatial arrangements. Intriguingly, in mammals the C-terminal region of mPer2, which is downstream of the casein kinase binding (CKB) domain, has been reported to be involved in directly binding to BMAL1 (Chen, 2009). Finally, the current findings strongly suggest that direct interactions between dPer and dClk/Cyc are required for dClk hyperphosphorylation. It is possible that some regulatory factors stay tightly bound to key clock repressors (such as Dbt to dPer) and thus require very close contact with central clock transcription factors to modulate them, whereas other factors are 'delivered' and establishing a high local concentration is sufficient to promote efficient transfer from the repressors to circadian-relevant transcription complexes (Sun, 2010).

A conserved transcriptional feedback loop underlies animal circadian rhythms. In Drosophila, the transcription factors Clock (Clk) and Cycle (Cyc) activate the transcription of direct target genes like period (per) and timeless (tim). They encode the proteins Per and Tim, respectively, which repress Clk/Cyc activity. Previous work indicates that repression is due to a direct Per-Clk/Cyc interaction as well as Clk/Cyc phosphorylation. This study describes the role of ubiquitin-specific protease 8 (USP8; FlyBase term, UBPY ortholog) in circadian transcriptional repression as well as the importance of Clk ubiquitylation in Clk/Cyc transcription activity. usp8 loss of function (RNAi) or expression of a dominant-negative form of the protein (USP8-DN) enhances Clk/Cyc transcriptional activity and alters fly locomotor activity rhythms. Clock protein and mRNA molecular oscillations are virtually absent within circadian neurons of USP8-DN flies. Furthermore, Clk ubiquitylation cycles robustly in wild-type flies and peaks coincident with maximal Clk/Cyc transcription. As USP8 interacts with Clk and expression of USP8-DN increases Clk ubiquitylation, the data indicate that USP8 deubiquitylates Clk, which down-regulates Clk/Cyc transcriptional activity. Taken together with the facts that usp8 mRNA cycles and that its transcription is activated directly by Clk/Cyc, USP8, like Per and Tim, contributes to the transcriptional feedback loop cycle that underlies circadian rhythms (Luo, 2012).

This study addressed the function of the deubiquitylating enzyme USP8 in Drosophila circadian rhythms. Although usp8 mRNA was one of the top cyclers identified in head RNA more than a decade ago, it was only recently identified as one of the few non-clock gene mRNAs that cycles in circadian neuron RNA as well as in head RNA. This revived interest in understanding its role in circadian rhythms. The data in this study indicate that USP8 deubiquitylates Clk, which likely contributes to inhibiting clock gene transcription at the appropriate trough times in the circadian cycle. This regulation appears particularly important for clock neurons because interference of USP8 function by either RNAi knockdown or expression of USP8-DN dramatically alters fly locomotor activity, ranging from long periods to complete arrhythmicity (Luo, 2012).

The expression of USP8-DN also had dramatic effects on the oscillations of both tim and per mRNAs and proteins. For example, expression of USP8-DN under the control of the tim driver increases the RNA levels of per, tim, and vri at almost all times of the circadian cycle. Effects on pre-mRNA were similar and included the usual trough times, from late night to early morning (CT22-CT6). The increased levels of mRNA presumably contribute to the increase in Per and Tim in head extracts at CT6-CT10, a time when there is no more than a trace visible in wild-type extracts. However, it is noted that per and tim RNAs and proteins are still cycling in the USP8-DN-expressing flies, in apparent conflict with the stronger behavioral phenotypes of these mutant flies. It is suspected that this inconsistency reflects at least in part the heterogeneity of fly head tissues; i.e., a substantial circadian contribution from the eyes might mask a quantitatively more important contribution of USP8 within circadian neurons. Indeed, expression of USP8-DN has a much more dramatic effect on the levels of Per and Tim at CT10 and tim mRNA at CT22 in PDF neurons, reflecting and perhaps even causing the long period or arrhythmicity of these flies (Luo, 2012).

Most USP family proteins stabilize their substrates by preventing proteasome-mediated degradation and therefore enhancing protein levels. Yet USP8-DN increases Clk activity without affecting Clk levels. The same conclusion results from the S2 cell experiments: RNAi knockdown of endogenous USP8 or transfection of USP8-DN enhanced Clk/Cyc transcription activity, which is still sensitive to Per repression. Based on the results from flies as well as S2 cells, USP8 probably functions as an inhibitor of Clk/Cyc transcriptional activation (Luo, 2012).

Inhibition of USP8 causes an increase in Clk ubiquitylation, mostly monoubiquitylation. This presumably reflects the interaction of Clk with USP8 in S2 cells and fly heads; i.e., the direct deubiquitylation of Clk. Interestingly, Clk ubiquitylation levels cycle and peak at ZT10-ZT14, when maximal transcription occurs. Moreover, expression of USP8-DN dramatically increased the level of Clk ubiquitylation at ZT10 and ZT22, especially at ZT22. Because Clk binding at the E-box of the tim promoter did not change dramatically between the two strains (and perhaps even in the opposite direction), it is likely that Clk monoubiquitylation does not enhance DNA binding, but rather potentiates transcriptional activity. Monoubiquitylation of a transcription factor has been shown to help recruit other transcription factors and even Pol II to a promoter (Luo, 2012).

Ubiquitylation may be mechanistically related to the 'black widow' model of transcription activation, in which transcription factors on chromatin are actively degraded by the proteasome. This may apply to mammalian BMAL1, the ortholog of Cyc, suggesting that the same turnover mechanism may be relevant to Clk/Cyc in flies. A maximally active chromatin-bound Clk may therefore be monoubiquitylated, which can then experience two inhibitory fates. It can be further ubiquitylated, perhaps directly on chromatin, which may then require proteasome-mediated degradation to allow replacement by another maximally active monoubiquitylated Clk, or it can be deubiquitylated by USP8. The former may predominate at times of maximal transcription so that rapid recycling of Clk/Cyc by the proteasome maintains maximal levels of monoubiquitylated Clk and maximal transcription rates. In the late night-early morning, deubiquitylation by USP8 may predominate and help minimize clock gene transcription. It is notable that these are circadian times when cycling usp8 mRNA levels are maximal. However, it is possible that regulation of an E3 ligase also contributes to the cycling of Clk ubiquitylation (Luo, 2012).

The importance of differential ubiquitylation is reinforced by the different ubiquitylated Clk-binding patterns on some clock genes; i.e., vri and pdp1. The fact that ubiquitylated Clk occurs preferentially close to the transcription start sites of these genes suggests that Clk monoubiquitylation may help recruit factors to drive transcription; Pol II or factors associated with transcription initiation are good candidates. In this view, the eye-specific vri and pdp1 Clk-binding sites with poor ubiquitylation may reflect Clk-binding sites without these cofactors, allowing deubiquitylation to predominate. The relatively poor Clk ubiquitylation at these sites may also indicate a relationship with other transcription factors. Put otherwise, deubiquitylated Clk may play a more modest role at these sites and partner with (unidentified) factors that contribute most of the transcriptional activation potential, perhaps within certain eye cell types (Luo, 2012).

Nonetheless, the fact that Clk deubiquitylation by USP8 appears maximal at the end of the transcriptional cycle suggests that deubiquitylated Clk is associated with changes in chromatin structure and/or transcription complexes at this time. Indeed, most Clk is sequestered by Per in an off-DNA inhibitory complex at the end of the cycle, suggesting that this complex contains substantial levels of deubiquitylated Clk. Per may enhance USP8 activity on Clk or inhibit Clk monoubiquitylation within the Per-Clk complex. Alternatively, Per may function strictly to inhibit DNA binding, suggesting that Clk monoubiquitylation and even deubiquitylation are predominantly on-DNA events (Luo, 2012).

It is interesting that the original analysis of cycling mRNAs within PDF neurons indicated that the circadian amplitudes of most clock gene mRNAs were dramatically enhanced compared with their amplitudes in head RNA (Kula-Eversole, 2010). This is consistent with the enhanced effect of USP8 inhibition of mRNA cycling in PDF neurons compared with the effects on head RNA, suggesting that USP8 function may contribute to this enhanced amplitude of clock gene cycling. A challenge for the future will be to understand how and why USP8 functions differentially within circadian neurons. It will also be important to integrate this goal with the ability to assay Clk/Cyc binding and even ubiquitylated Clk binding as a function of circadian time within PDF neurons (Luo, 2012).

GENE STRUCTURE

Within a Drosophila Clock intron, a second ORF is found with significant similarity to the mammalian gene EB1, implicated in binding the adenomatosis polyposis coli (APC) C terminus (Allada, 1998).

PROTEIN STRUCTURE

Amino Acids - 1015 (Allada, 1998); 1023 (Darlington, 1998)

Structural Domains

Sequencing of multiple cDNAs indicates one open reading frame. The ORF has at least two forms: one corresponds to the full-length protein of 1015 amino acids and the other to a protein missing the bHLH and PAS A regions. Several transcript forms are found, none of which demonstrate robust circadian oscillations. The full-length Drosophila Clock protein contains all of the known subregions of mouse Clock, including bHLH, PAS A, PAS B, and prominent Q-rich activation domain (Allada, 1998).

Alignment of the full-length Drosophila Clock protein with mouse Clock reveals 35% identity over the entire overlap (>800 amino acids). Drosophila Clock also shows substantial sequence similarity to the mouse and human bHLH-PAS proteins NPAS2/MOP4, but the latter has no polyQ regions. There is no functional evidence, however, linking NPAS2/MOP4 to circadian rhythms. The sequence identity of Drosophila and Mouse Clock proteins is even more impressive in the three subregions where one can infer a biochemical function. The bHLH domains, involved in DNA binding and protein dimerization, have 71% similarity and 60% identity. The basic region, involved in sequence-specific DNA contacts, is remarkably conserved with 11 out of 13 amino acids being identical; this suggests that the two proteins bind to similar if not identical DNA targets. In fact, 6 out of 9 amino acids are identical to a consensus generated for bHLH proteins that bind the CAC/GTG E box half-site, including the critical R residue at position 15 (Allada, 1998).

The PAS region, implicated in protein dimerization, is also strikingly conserved between the insect and murine genes. The PAS B repeat and the region within PAS just C-terminal to PAS B are particularly conserved; 79% identical and 91% similar over a span of 107 amino acids (amino acids 262-368). The conservation of PAS and its demonstrated role in dimerization suggest that Drosophila Clock and mouse Clock may have conserved heterodimeric partners. Indeed, it appears that another Drosophila clock gene, cycle, encodes the relevant bHLH-PAS partner and that the same heterodimeric complex is functionally relevant in both systems. The third conserved region is the glutamine (Q)-rich C terminus of the protein. Glutamine-rich regions, especially polyglutamine repeats, are known to function in transcriptional activation (Allada, 1998).